Selenium is an essential micronutrient in the diet of humans and other mammals and many health benefits have been ascribed to this element including preventing cancer, heart disease and other cardiovascular and muscle disorders, inhibiting viral expression, delaying the progression of AIDS in HIV positive patients, slowing the aging process, and having roles in mammalian development, male reproduction and immune function. We proposed previously that the health benefits of selenium are due in large part to the presence of selenium in selenoproteins as the selenium-containing amino acid, selenocysteine (Sec), and since little was known about how Sec was biosynthesized, we undertook a project to elucidate how this amino acid, which is the 21st amino acid in the genetic code, was synthesized and to identify and characterize each of the components involved in its pathway. We previously established the biosynthetic pathway of Sec in eukaryotes and archaea and are now focusing our attention on the two Sec tRNA isoforms that we have shown are responsible for the synthesis of the two subclasses of selenoproteins designated housekeeping and stress-related selneoproteins;and on the methylase, designated Um34 methylase, that synthesizes the methyl group at the 2-O-position on the ribosyl moiety at position 34 of Sec tRNA. In the past year, we have confirmed that addition of Um34 to the isoform, 5-methylcarobxymethyluridine (mcmU), to form 5-methylcarboxymethyl-5-,2-O-methyluridine (mcmUm) requires that mcmU is aminoacylated with Sec, i.e., that the substrate for the methylase (designated Um34 methylase) which carries out this reaction is selenocysteyl-tRNA. These observations were established by generating a Sec tRNA encoding a mutation at position 74 which is the discriminator base in all tRNAs wherein the resulting mutant tRNA cannot be aminoacylated. Microinjection of the discriminator base mutant Sec tRNA into Xenopus oocytes, overnight incubation, isolation and analysis of the resulting minor base modifications demonstrated that all minor bases were present except Um34 demonstrating that the non-aminoacylated Sec tRNA could not serve as a substrate for Um34 methylase. Furthermore, co-microinjection of a potent inhibitor of seryl-tRNA synthetase, SB-217452, and the wild type Sec tRNA also showed that all minor bases were synthesized on the tRNA with the exception of Um34, again showing the non-aminoacylated Sec tRNA could not serve as a substrate for Um34 methylase. Using a bioinformatics approach, we tentatively identified two possible candidates as Um34 methylase in human and mouse cell lines. Cloning both purported methylases into an expression vector, and expressing and characterizing them showed that one was indeed Um34 methylase. Transfecting mouse and human cell lines with a knock-in vector encoding Um34 methylase demonstrated that over-expression of Um34 methylase resulted in over-expression of stress-related selenoproteins. Furthermore, transfecting mouse and human cell lines with a vector targeting the removal of Um34 methylase demonstrated that the loss of the methylase resulted in down-regulation in the expression of stress-related selenoproteins. We are currently characterizing Um34 methylase in vitro experiments. We initially found that cysteine could replace Sec in thioredoxin reductase 1 (TR1) and have expanded these studies extensively in the past year. Although cysteine is normally inserted into proteins in response to UGC and UGU codons, we observed that supplementation of NIH 3T3 cells with thiophosphate resulted in the targeted insertion of cysteine at the Sec, UGA codon in TR1. Cysteine was synthesized by Sec synthase on Sec tRNA, and importantly, the insertion was dependent on the Sec insertion sequence (SECIS) element in the 3- UTR of TR1 mRNA. The SECIS element is an absolute requirement for Sec insertion into all selenoproteins. The substrate for the reaction that synthesizes cysteine on Sec tRNA in place of Sec is thiophosphate that is synthesized by selenophosphate synthetase 2 from ATP and sulfide wherein the newly synthesized cysteine in turn is donated to phosphoseryl-tRNA(Sec) to generate Cys-tRNA(Sec). Cysteine was also found to be inserted in vivo at UGA codons in mammalian thioredoxin reductases 1 and 3, and this process was regulated by dietary selenium and availability of thiophosphate. Cysteine was found at 10% of the Sec levels in mouse liver TR1 when the mice were maintained on a diet with normal amounts of selenium and at 50% in liver TR1 of mice maintained on a selenium deficient diet. The data reveal a novel Sec machinery based mechanism for biosynthesis and insertion of cysteine into protein at UGA codons and suggest new biological functions for thiophosphate and sulfide in mammals. In addition, we are examining the replacement of Sec by cysteine and other amino acids in cancer cells and in cells treated with antibiotics. Cancer cells have shown a high percentage of dehydroalanine in place of Sec in TR1. These studies have only just gotten underway and will be reported on in greater depth in next years report.